SYNTHESIS AND BIOLOGICAL ACTIVITIES OF SOME NEW HETEROCYCLES CONTAINING NITROGEN AND SULPHUR

SYNTHESIS AND BIOLOGICAL ACTIVITIES OF SOME NEW HETEROCYCLES CONTAINING NITROGEN AND SULPHUR

Md. Akram ^*, Abdul Sayeed and Syed Yousuf Hussain

MESCO College of Pharmacy Hyderabad (T.S), Andhra Pradesh, India.

ABSTRACT: The literature survey on 1,2,4-Triazoles and chalcone derivatives revealed that they  are  endowed  with  wide  variety  of  biological  activities. During the present investigation newer series of chalcone derivatives S-4-(isonicotinamido)-5-(phenoxymethyl) - 4H-1,2,4 triazole-3-yl-3(-4-chlorophenyl) propanethioate (7a-7f) were synthesized by coupling  suitably  modified 1,2,4-Triazole nucleus with appropriate aryl aldehydes. The structures of the newly synthesized compounds were established by FTIR, ¹HNMR and Mass spectral analysis. All the compounds have been evaluated for in vitro antibacterial, antifungal and antitubercular activities. The compounds exhibited weak antibacterial activity only at higher concentrations. Whereas,  the  antifungal  activity  results  showed  that  few  of  the compounds  were  able to show moderate antifungal potency. The fungi used in the anti-microbial study were found to resistant to   the other compounds. The antitubercuar activity results were encouranging as the few compounds were able to possess growth inhibiting properties against the mycobacterium tested i.e the compounds 7b, 7e and 7f possessed the MIC as low as 6.25µg/ml. Even though the compounds failed to possess anti-bacterial and antifungal activity, it is quite interesting to note that most of the compounds were found to be promising biological agents as antitubercular.

Chalcone Derivatives, Antibacterial, Antifungal, Antitubercular

INTRODUCTION: Heterocyclic compound is one which possesses a cyclic structure with at least two different kinds of atoms in ring. One of which is carbon, and can be aliphatic or aromatic. A hetrocyclic compound usually possesses a stable ring structure this does not readily hydrolyze or depolymerize. It is obvious that the number and variety of hetrocyclic compound with reported antimicrobial activity is extensive with respect to structure and application.  Heterocyclic compounds exhibited remarkable pharmacological activities.

The hetrocyclic compounds containing pyrimidine,  pyridine, and piperazine nucleus have wide range of therapeutic activity such as antitubercular, anticancer, antihelmintic, antioxidant and antimicrobial activities is also believed that the presence of N-C=S linkage is responsible for the amoebicidal, anticonvulsant, fungicidal and antiviral activities ^1-4.

In this direction, the work is being pursued to investigate the antimicrobial activity of some heterocyclic compounds prepared in our laboratory. Various piperazine nucleus containing derivatives have been synthesized. All the synthesized derivatives show better anti bacterial and anti fungal activity against various strains of bacteria and fungai viz.  Escherichia  coli,  Pseudomonas  aeruginosa,   Klebsiella  pneumonaie.,  Aspergillus niger,  Microsporum  gypseum.

After  the  antimicrobial  studies, it   were  found  that hetrocyclic   Compound   showed   excellent   activity   against   the   bacterial   strain   of Pseudomonas aeruginosa and compound showed very good activity against the fungal strain of  Aspergillus niger.

Incorporation  of oxygen,  nitrogen,  sulphur,  or  an  atom  of a related  element  into  an organic ring structure in place of a carbon atom gives rise to a heterocyclic compound. Since the heterocyclic atom must form more than one bond in order to be incorporated into a ring structure, halogens do not form heterocyclic compounds although they may be substituents on a heterocyclic ring structure. Heterocyclic compounds, like polycyclic ring compounds, are usually known by non-systematic names. They may be either simple aromatic rings or non-aromatic rings.  Some examples are pyridine, pyrrole, furan and thiophene.

Many heterocyclic compounds are biosynthesized by plants and animals and are biologically active. Some heterocycles are fundamental to life, such as haem derivatives in blood and the chlorophylls are essential for photosynthesis. Similarly, the paired bases found in RNA and DNA are heterocycles, as are the sugars that in combination with phosphates provide the backbones and determine the topology of these nucleic acids. The biological properties of heterocycles in general make them one of the prime interests of the pharmaceutical and biotechnology industries.

Triazoles: In five-membered ring systems, the presence of three nitrogen atoms defines a interesting class of compounds, the triazoles. These may be of two structural types, the 1,2,3 triazoles or v-triazole (1) and 1,2,4-triazoles or s-triazoles (2).

The name triazole was first given to the carbon nitrogen ring system C2H3N3 by Bladin, who described its derivatives in 1885. Later on because of various applications, triazoles took special attention particularly by the chemical industry. Different chemical reactions show that 1,2,4-triazole is an extremely stable nucleus, which can be regarded as aromatic in nature, stabilized by resonance as shown below.

4,5-Disubstituted-1,2,4-triazole-3-thiones: Triazoles  having  a  mercapto  group  at  C-3  (3)  constitute  a  special  class  of compounds among the substituted 1,2,4-triazoles because of the tautomerism exhibited by them.  The labile hydrogen atom may be attached to either nitrogen or sulfur atom. Previous  studies  of  IR,  NMR  and  UV  spectra  have  provided  strong  evidence  that mercaptotriazoles are present predominantly in the thione form (4).

Due to lability of hydrogen, both the S-substitution and N-substitution can be carried  out  by  changing  the  reaction  to  electron  deficient  alkenes  and  alkynes,  ring opening  of oxiranes and acylation.

At low temperatures, oxidative chlorination of mercaptotriazoes is an excellent method of obtaining the corresponding sulfonyl chloride which can be converted to sulfonamide. The mercapto group can be eliminated using reagents like Raney nickel, dilute nitric acid or hydrogen peroxide. Mercapto groups are easily converted into their methylethers  with  sodium hydroxide and methyliodide and these  ethers  have  little  tendency  to  lose  methanethiol  on  standing. Some important reactions of thiols are shown in below.

5-(Substituted)-4-amino-1,2,4-triazole-3-thiones: Among the substituted triazoles, aminotriazoles constitute class because of their widespread biological activities eg. 3-aminotriazole, better known as amizol was the first triazole which was used as an important herbicide. Aminotriazoles may have a C-amino group or an N-amino group. C-aminotriazoles behave as normal aromatic amines and can be diazotized in aqueos mineral acid with nitrous acid, forming diazonium salts which couple with aromatic bases. The detection of 3-amino-1,2,4-triazole in plant extracts depends

on this property. Both C- and N-aminotriazoles undergo other characteristic reactions of a primary amine. The amino group of 4-amino-1,2,4-(4H)-triazole reacts with aldehydes forming the  corresponding Schiff base.  The Schiff bases obtained from 5-nitrofufural and 5- nitrofurylacylic aldehyde compare favorably with furacin (5-nitrofurfural semicarbazone) in bacteriostatic properties (Eq. 1.1). As expected, ethyl chloroformate readily forms ethyl N-(1,2,4-(4H)-triazol-4-yl)carbamate with 4-amino-(4H)-1,2,4-triazole (Eq. 1.2).

Biological Activities of 1,2,4-Triazoles: 4,5-disubstituted-2,4-dihydro-3H-1, 2, 4 – triazole - 3-thiones   and   their   derivatives have gained a  lot of interest in the last decade due to their biological, industrial and agricultural importance. A well known example is that of fluconazole, a broad spectrum antifungal agent for the treatment of superficial and systemic infections. Recently it has been investigated that 1,2,4-triazoles are associated with a  variety of pharmacological activities  such  as  antifungal,  diuretic,   antibacterial,  hypoglycemic,   antitubercular, antidepressant, antiamoebic, antibiotic, anti-infalmmatory, anti-carcinogenic, hypnotic, sedatives,  plant  growth  regulators  and  insecticidal. Some pyridyl and pyrimidyl substituted mercaptotriaoles (5) have been reported to exhibit antithyroid activity.

N-aminomercaptotriazoles (6) have been reported to show significant bactericidal and fungicidal activity. Some disubstituted mercaptotriazoles (7) and (8) have been tested in vitro against a number of fungi and they showed moderate to good activity.

The highest activity against all species was shown by (8) (R=4-IC₆H₄). Acylation of some aminotriazoles gives triazoleamides (9) which specifically inhibited rubella virus 4- Amino-l,2,4-triazoles and their derivatives (10) and (11) have been prepared and found to possess bactericidal and/or fungicidal activity. Certain 4-alkylsubstituted triazoles (12) have been reported to inhibit reserpine-induced ptosis in mice with an ED50 of 0.27 mg/kg. Certain substituted 5-(4-pyridyl)-3-mercapto-l,2,4-triazoles  (13) have been used as additives.

1,2,4-Triazolcs and condensed triazole systems have found considerable use in photographic  industry.  1,2,4-triazole  derivatives  e.g.  N,N'-bi(5-methyl-l,2,4-triazol-3- yl) formamidines are capable of  inhibiting fog formation in silver halide emulsions by incorporating in the emulsions. The addition of 5-mercaptotriazole or their soluble salts to the emulsion after development produces a greater maximum image density and prevents bronzing effect. The sodium salt of a sulfonated derivative possesses good detergent action e.g. N-benzylated aminotriazole (14) have useful properties in inhibiting the acid fading of dyestuff. Some 4-(phenylureido)-1, 2,4-triazole derivatives have been used   as defoliants   e.g. (15;50.0 wt. parts) when formulated with sodium- dodecylbenzenesulfonate 3.0,  sodium-ligninsulfonate 2.0, SiO: 15.0 and clay 30.0 wt. parts gives 50% wettable powder and it causes 50% defoliation of cotton at 0.5 kg/ha, Vs 20%with(BuS)PO.

A new series of condensation polymers polyaminotriazoles have been prepared, which are similar to nylon.  These are fibre forming and be melt spun to  give filaments which after drawing, possess high strength and good affinity for dyestuffs.

These polyaminotriazoles are condensation polymers which are produced by heating dihydrazides in presence of small amount of hydrazine. The name refers to the rearranging

linkage,   the   4-amino-l,2,4-triazole  ring  (Eq,  1.3).  Some  1,2,4-triazole derivatives  (16)  and  (17)  have   been   used  as  stabilizers  for  chlorine-containing thermoplastic polymers.

Objectives:

Need for the study: The chemistry  of  heterocyclic  compounds  is  the  field  in  the  organic chemistry which is being explored continuously. The importance of 1,2,4-triazole derivatives  lies  in  the  field  that,  these  derivatives  represent  one  of  the  most biologically active classes of compounds, possessing a wide range of therapeutic properties ^5-6. Chalcones  constitute  an  important  family  of  substances  belonging  to flavonoids,  a  large  group  of  natural  and  synthetic  products  with  interesting physicochemical  properties,  biological  activity  and  structural  characteristics.

Chalcones are highly reactive substances of varied nature, and they experience chemical and physical transformations of great importance. These are characterized with diverse biological activities, among which anti- inflammatory, anti-malarial, anti-protozoal, anti bacterial, cytotoxic, anti- mitotic, anti-viral, anti-inflammatory, antileishmanial, and antitubercular activity.

The above  mentioned  reports  encouraged  us  to  modify  1,2,4-triazole scaffold into  various bioactive structures. Hence, in the present study we have synthesised  some  new   derivatives  of  1,2,4-triazole  and  evaluate  them  for antimicrobial and  antitubercular activities.

Chalcones have been studied extensively because of their ready accessibility, diverse chemical reactivity and broad spectrum of biological activities.

The present investigation includes the following;

1. Preparation of  3-phenoxmethyl-4-N-(pyridyl  carboxamido)-  5-mercapto  1,2,4-triazole by following known me
2. Conversion of the above compound to form S-4 (isonicotinamido)-5- (phenoxymethyl) - 4H -1, 2, 4-triazol-3-yl ethanethioate.
3. The reaction of the above compound with substituted benzaldehydes in presence of 2% NaOH to yield the title derivative
4. Chemical characterization of the newly synthesised compounds by IR, ¹HNMR and
5. Mass spectral data.
6. To screen the synthesised compounds for the anti-microbial and antitubercular activity.

MATERIAL AND METHODS: The  entire  chemicals  used  were  procured  from  Qualingens,  Himedia  and  Loba- chemicals.  Purity of starting materials used for reaction was confirmed by checking their melting point or boiling point and by thin layer chromatography.

SCHEME: 1

(B). Synthetic Studies:

Step 1: Preparation of 2-Phenoxyacetohydrazide (3):-

The acid (1) (0.1 mole) and ethanol (50 ml) were taken with a few drops conc. H2SO4 and was refluxed for 6 hours. The reaction mixture was concentrated by distilling off the excess of ethanol under reduced pressure. The ester (2) obtained was used for the preparation of hydrazide directly.

The ester (2) (0.1 mole) was dissolved in appropriate quantity of ethanol and to this hydrazine hydrate (0.1 mole) was added. The reaction mixture was refluxed for a period of 12-18 hours. Excess of ethanol was distilled off under reduced pressure. It was  then poured  into  ice cold  water  and  the  solid  obtained  was  filtered. It was recrystallized from ethanol.

Step 2: Preparation of potassium-2-(2-phenoxyacetyl) hydrazinecarbodithioate (4):-

To a solution of potassium hydroxide (KOH) (0.15 mole) in absolute ethanol (125 ml), 2-phenoxyacetohydrazide (3) (0.1 mole) and carbon disulphide (CS2) (0.15 mole) were added and the mixture was agitated for 16 hours. To the resulting solution, anhydrous ether (250 ml) was added and the precipitated product (4) was collected by filtration, washed with ether and dried under vacuum at 65ºC. This potassium salt (4) was used in the next step without further purification.

Step 3: Preparation of N-(5-mercapto-3-(phenoxymethyl) - 4H-1, 2, 4-triazol-4- yl)     iso nicotinamide (5) :-

A suspension of the potassium salt (4) (0.1 mole), Isonicotinic acid hydrazide (INH) (0.1 mole) and water (5 ml) were heated under reflux for 6 hours and hydrogen sulphide (H₂S) gas was evolved and clear solution was resulted. The dilution of reaction mixture with cold water (50 ml) and subsequent acidification with dilute hydrochloric acid (HCl) gives the triazole (5), which was filtered, washed with water and recrystallized from aqueous ethanol.

Step 4: Preparation S-{5-(phenoxymethyl)-4-[(pyridin-4-ylcarbonyl) amino]-4H-1,2,4-triazol-3-yl} ethanethioate (6) :-

The mixture of triazole (5) (0.01 mole) and (0.01mole)   of potassium carbonate is taken in a RBF to this add 50ml of actone and stir the mixture on magnetic stirrer for 10min. Then add 0.01mole acetyl chloride by dropwise using funnel. After complete addition reflux the reaction mixture for about 4hours cool the reaction mixture and add 100ml of water filter and wash with water.

Step 5: Preparation of S-4-(isonicotinamido)-5-(phenoxymethyl 4H-1, 2, 4 triazole- 3-yl-3 (substituted phenyl) propanethioate (7a-7f) :-

To a mixture of equimolar quantities of triazole and aromatic aldehyde (0.005 mole)  in ethanol(25ml) was added and stirred for 10hours at room temperature. Then it was refluxed for 6hrs on water bath. The excess of solvent was removed under reduced pressure it was poured into ice-cold water. The solid thus separated was filtered dried and recrystallized from   ethanol. The physical data of compounds (7a-7f) are summarized in Table 1.

TABLE 1: PHYSICAL DATA OF (E)-S-4-(ISONICOTINAMIDO)-5-(PHENOXYMETHYL)-4H 1,2,4-TRIAZOL-3-YL 3-(SUBSTITUTED PHENYL)PROP-2- ENETHIOATE(7A-7F)

All melting points were uncorrected         Mobile phase: Acetone: Benzene (1:1)

Biological activities:

Anti-Tubercular Activity: All  the  synthesised  1,2,4-  triazole  derivatives  have  been  evaluated  for  Anti- tubercular activity against Mycobacterium tuberculosis H37  Rv using Microplate alamar blue dye assay(MABA) .The minimum inhibitory concentration(MIC) was determined for each  of  the  sample.  The  first  line  antitubercular  drug  INH  was  used  as  a  refrence standard. The resulte are tabulated in table no-4 and graphically depicted in Fig. 1.

Anti tubercular activity using Alamar Blue Dye Method: The  anti-mycobacterial   activity  of  the  title  compounds  were  assessed  against Mycobacterium  tuberculosis H37  Rv using Microplate Alamar Blue Assay (MABA) method. This method is non-toxic, uses a thermally stable reagent and shows good correlation with proportional and BACTEC radiometric method. Briefly, 200µl of sterile deionzed water was

added to all outer perimeter wells of sterile 96 wells plate to minimized evaporation of medium in the test wells during incubation. The 96 wells plate received 100 µl of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on plate. The final drug concentrations tested were 100 to 0.2µg/ml. Plates were covered and sealed with parafilm and incubated at 37ºC for seven days. After this time, 25µl of freshly prepared 1:1 mixture of Alamar Blue reagent and 10% tween 80 was added to the plate and incubated for 24 hrs.  A blue color in the well was interpreted as no bacterial growth and pink color was scored as growth. The MIC is defined as lowest drug concentration which prevented the color change from blue to pink.

Antimicrobial activity: ^7-8 In  general,  any  compound  or  drug  which  inhibits  the  growth  or  causes  the death of micro-organisms is known as antimicrobial agent. Any drug which inhibits   the growth of bacteria or fungi, it is said to possess bacteriostatic and fungi static activity respectively.

If  it  kills  the  bacteria  or  fungi,  it  is  said  to  possess  bactericidal  and fungicidal activity.  In- vitro tests are used as screening procedure for new agents and for testing the susceptibility of individual isolates from infections to determine which of the available drug might be useful  therapeutically important factors for the antimicrobial activity  and  size  of  the  inoculums,  metabolic  state  of  organisms,  pH,  temperature, duration  of  interaction,  concentration  of  the  inhibitors  and   presence  of  interfering substance. Sensitivity testing is done to determine the range of microorganisms that are susceptible to the compound under specified conditions. It can be done by cup-plate method. This method is suitable for the organisms that grow well overnight such as most of the common aerobes and facultative anaerobes and rapidly growing fungi. Several forms of disk diffusion methods have been advocated.

Biological evaluation involves testing of microbial susceptibility to chemotherapeutic agents. Determination of antimicrobial effectiveness against pathogens is essential for therapy.  Testing can show the efficiency of antimicrobial against a pathogen and give an estimate of proper therapeutic dose. The idea of the effectiveness of a chemotherapeutic agent against a specific pathogen can be obtained from the minimum inhibitory concentration (MIC). The MIC is the lowest concentration of the drug that can prevent   the   growth   of the pathogen.

The important factors to be considered in the testing of the antimicrobial activity are as follows:

1. Type of test organism.

1. Temperature and time of incubation.
2. Composition and pH of culture.
3. Inoculums concentration.

1, 2, 4, triazole derivatives described in the literature are known for their antimicrobial activity. Hence, in the present study, substituted triazole derivatives synthesized were screened for their antibacterial as well as antifungal activity using various bacterial strains as well as fungal strains.

Evaluation of Antibacterial Activity: Antibacterial activity was determined based on the in vitro activity in pure cultures. In vitro susceptibility test were done by the cup-plate method. The antibacterial activity of formazan derivatives was evaluated by cup-plate method against the strains of common   pathogens; gram negative organisms Escherichia coli, Pseudomonas aeruginosa and Gram positive organisms Staphylococcus aureus Bacillus subtilis. Ciprofloxacin is used as a standard drug.

MATERIAL AND METHODS:

Test organisms (bacteria)

Bacillus subtilis-   Gram positive bacteria

Staphylococcus aureus- Gram positive bacteria

Escherichia coli- Gram negative bacteria

Pseudomonas aeruginosa- Gram negative bacteria

All the synthesized compounds were screened for antibacterial activity against the above mentioned strains by cup-plate method. The following materials were used for the testing.

1. Nutrient agar.
2. Sterilized petridish, pipettes and beaker
3. Sterilized tuberculin syringe
4. 18-24 hr old growth culture in nutrient br
5. Sterilized test tubes containing solution of test compounds in desired concentration

Preparation of Nutrient agar media

Nutrient agar (40g), bacteriological peptone (1g), beef extract (5g) and sodium chloride (5g) were dissolved in distilled water (1000 ml). The pH of the solution was adjusted to 7 to 7.4 by using sodium hydroxide solution (40%, approximately 0.25 ml for 100 ml of nutrient broth) and then sterilized for 30 min. at 15 lbs pressure in an autoclave.

Preparation of sub culture: One day prior  to  test  the  microorganisms  were  inoculated  into  the  sterilized nutrient broth and incubated at 37ºC for 24 hr on the day of testing the organisms were sub-cultured  into  sterile  nutrient  broth. After incubating for 3hr, the growth thus obtained was used as inoculums for the test.

Sterilization of media and glass wares: The media  used  in  the  present  study,  nutrient  agar  and  nutrient  broth  were sterilized in a conical flask of suitable capacity by autoclaving the same at 15 lbs pressure for 20 min. The petridishes, test tubes and pipettes were sterilized by employing hot air oven at 160ºC for 1 hr.

Preparation of solution of test compound: The test compound (10 mg each) was dissolved in freshly distilled DMF (10 ml) in serially labeled sterile test tubes, thus giving a final concentration of 50µg/0.1ml,100µg/0.1ml.

Preparation of standard solution: The standard compound ciprofloxacin (10 mg) was dissolved in freshly distilled DMF (10 ml) in serially labeled sterile test tubes, thus giving a final concentration of 50µgm/0.1ml, 100µgm/0.1ml.

Method of Testing:

Cup-plate Method: This method depends on the diffusion of an antibacterial agent from a cavity through the solidified agar layer in a petridish to an extent such that growth of the added  microorganisms is prevented entirely in a circular area or zone around the cavity containing a solution  of test compounds. About 15-20 ml of molten nutrient agar was poured into each of the sterile petridishes. The cups were made by scooping out nutrient agar with a sterile cork borer. The agar plates so prepared were divided into different set and each set of the plates were inoculated with the suspension of particular organism by spread plate technique.

The cups of inoculated plates were then filled with 0.1 ml of the test solution; the plates were then incubated at 37ºC for 24 hours. The zone of inhibition (diameter in mm) developed, if any, was then measured for the particular compound with each organism. The solvent DMF was used as negative-control to know the activity of the solvent.

The results of antibacterial testing are summarized in the following Table 3. The antibacterial activity of the tested compounds are then compared with that of standard drug used i.e. Ciprofloxacin.

Antifungal activity: The antifungal activity of formazan derivatives was carried out by Cup-plate method in comparison with that of standard antifungal drug clotrimazole. The fungi cultures used were Candida albicans and Aspergillus niger.

MATERIAL AND METHODS:

Cup-plate method: Antifungal activity of the test compounds was assessed against the above strains of fungi by disc-diffusion method. The following materials were used:

1. Sabourauds agar and tuberculin syringes with needle
2. Sterilized petri-dishes and pipettes of 1 ml and 0.2 ml.
3. 16-18 hr old cultures grown in Sabourauds broth.
4. Sterilized test tubes for preparation of solution of the test compounds in desired concentrati

Preparation of media:

Sabourauds agar: Bacteriological peptone (1 g) and glucose (4 g) were dissolved in distilled water (100 ml) and filtered.  Agar powder (2 g) was added and sterilized for 30 min at 15 lbs pressure.

Preparation of sub cultures: One day prior to the test, inoculation of the microorganisms (Candida albicans and Aspergillus niger) was made in sabourauds broth and incubated at 37ºC for 18 hr.

Sterilization of media and glass wares.

The media used in the present study was sterilized in conical flask of suitable capacity by autoclaving at 15 lbs pressure for about 20 min. The petridish, test tubes and pipettes were sterilized in hot air oven at 160ºC for one hour.

Preparation of solution:

1. 1. Compounds: 10 mg of each test compounds was dissolved in 10 ml of DMF in serially and suitably labeled in sterile test tubes thus giving a final concentration of 50µg/0.1ml, 100µg/0.1ml.
2. Clotrimazole: 10 mg of the clotrimazole was dissolved in 10 ml of DMF (dimethyl formamide) to get a concentration of 50µg/0.1ml, 100µg /0.1ml.

Method of Testing:

Cup-plate Method: This method depends on the diffusion of an antibacterial from a cavity through the solidified agar layer in a petridish to an extent such that growth of the added microorganisms is prevented entirely in a circular area or zone around the cavity containing a solution of test compounds. About 15-20 ml of molten nutrient agar was poured into each of the sterile petridishes. The cups were made by scooping out nutrient agar with a sterile cork borer. The agar plates so prepared were divided into different set and each set of the plates were inoculated with the suspension of particular organism by spread plate technique.

The cups of inoculated plates were then filled with 0.1 ml of the test solution; the plates were then incubated at 37ºC for 24 hours. The zone of inhibition (diameter in mm) developed, if any, was then measured for the particular compound with each organism. The solvent DMF was used as negative-control to know the activity of the solvent. The results of antifungal testing are summarized in the following Table 4. The anti-fungal activity of the tested compounds are then compared with that of standard drug used i.e. Clotrimazole.

RESULTS AND DISCUSSION: During the present investigation, the title compounds 1-(4-(isonicotinamido)-5- (phenoxymethyl)-4H-1,2,4-triazol - 3 –y l) - 3, 5 - diphenylformazan  were  synthesized  as  per the scheme described.

By  analysis  of  spectral  data  of  the  representative  compounds  reveals  the successful  information of the synthesized substituted formazan derivatives possessing 1,2,4-Triazole scaffold. All the synthesized derivatives remitted in the products with good yield. Purity of all the synthesized compounds were checked by their melting point as well as TLC. The structure of synthesized compounds has been established and confirmed by spectral data obtained viz, FT-IR, ¹HNMR and Mass. All the synthesized compounds were evaluated for in vitro antimicrobial and some of the selected compounds for anti- cancer activities. Few of the selected compounds were also screened for their in vivo analgesic and anti-inflammatory activities.

Biological Activities:

Antimicrobial Evaluation: 1,2,4  triazole   derivatives   described   in   the   literature   are   known   for   their antimicrobial  activity; hence, in the present study also substituted triazole derivatives synthesized  were screened  for  their antibacterial  as  well  as  antifungal  activity using various bacterial strains as well as fungal strains. In vitro antimicrobial study was carried out by Cup-plate method. All the compounds were screened for antimicrobial activity at different concentration levels against the bacterial strains Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis. Antifungal activity was tested on Sabouraud dextrose agar (Himedia) plates (26ºC, 48-72h) by Cup-plate method against Candida albicans and Aspergillus niger at different concentration levels. Ciprofloxacin and Clotrimazole were used  as  reference  standards  for  comparison  of  antibacterial  and  antifungal  activity respectively. The results are tabulated in Table 2 and Table 3.

TABLE 2: ANTIBACTERIAL ACTIVITY OF (E)-S-4-(ISONICOTINAMIDO)-5-(PHENOXYMETHYL)- 4H-1, 2, 4-TRIAZOL-3-YL 3-(SUBSTITUTEDPHENYL)PROP-2- ENETHIOATE. (7A - 7F);

TABLE 3: ANTIFUNGAL ACTIVITY OF (E)-S-4-(ISONICOTINAMIDO)-5-(PHENOXYMETHYL)-4H-1,2,4-TRIAZOL-3-YL 3-(SUBSTITUTEDPHENYL)PROP-2- ENETHIOATE. (7A-7F)

Antitubercular Activity Studies: The literature  survey  has  revealed  that,  the  moieties  containing  1,2,4- triazole  nucleus  have  shown  to  posses  antitubercular  activity. Hence, in the present investigation all the synthesised 1,2,4-triazole derivatives (7a-7f) have been evaluated for  antitubercular activity  against  mycobacerium  tuberculosis H37Rv following microplate alamar blue assay  method. MIC was determined for each of the compound and first line antitubercular drug INH was used as the refrence standard. The  results  of  the  study  are  tabulated  in  Table  4  and graphically depicted in Fig. 1.

TABLE 4: ANTITUBERCULAR ACTIVITY OF AGAINST MYCOBACERIUM TUBERCULOSIS H37RV

FIG. 1: ANTITUBERCULAR ACTIVITY OF S-4-(ISONICOTINAMIDO)-5-(PHENOXYMETHYL)- 4 H- 1,2,4 TRIAZOLE - 3 - YL- 3 (SUBSTITUTED PHENYL) PRO PANETHIOATE (7A-7F) AGAINST MYCOBACERIUM TUBERCULOSIS H37RV

Antibacterial and Antifungal Studies: All the synthesized compounds were evaluated for antibacterial activity among which only the compound 7a has shown weak activity.

Whereas the other compounds failed to show growth inhibiting properties even at higher concentration levels tested.

The antifungal studies revealed that the compounds 7a-7d were able to show moderate activity when compared to that of standard clotrimazole.

Rest of the compounds have failed to show the potency as antifungal.

Antitubercular Activity Studies: The results of antitubercular activity are encouranging because

the compounds 7b,7e and 7f  have shown to possess the lowest MIC of 6.25µg/ml.

The other compounds 7a, 7d and 7c evaluated for antitubercular activity have also exhibited significant potency by possessing MIC of 12.5,25 and 50µg/ml respectively.

SUMMARY AND CONCLUSION: The development of new antimicrobial therapeutic agent with enhanced potency, high  selectivity  and  reduced  toxicity  is  a  constant  process  in  medicinal  chemistry. Exhaustive literature survey on 1,2,4-Triazoles and chalcone derivatives revealed that they possess wide range of biological properties.

It has been observed that combination of biologically active moieties in to one molecule and synthesizing newer moieties have been the methods of research. Based on these observations,  during the  present investigation  newer  chalcone  derivatives  were synthesized by coupling suitably modified   1,2,4-Triazoles nucleus  and evaluated them for in vitro antimicrobial and antitubercular activities.

Synthesis  of  all  the  chalcone derivatives  by  the  procedure  described  in methodology, resulted in products with good yields.  All the reactions were carried out under prescribed laboratory conditions. The products were purified by recrystallization.

Melting points were determined in open capillary method and were uncorrected. The structure of newly synthesised compounds was established by IR, ¹HNMR and Mass spectral studies.

The in vitro antimicrobial activity results showed that all the compounds failed to possess antibacterial properties. However, most of the compounds have shown to exhibit properties in inhibiting the growth of the fungal organisms.

The results of antitubercular activity studies were encouranging as most of the compounds have exhibited excellent growth inhibiting properties against   the mycobacterium tested.

All the above results only indicate that, the compounds of the above type has emerged as promising antitubecular agents. Further study on the above type of moiety is quite reasonable and desirable Slight modification in the above type of moieties may lead to the discovery of new useful therapeutic agents.

Given more attention on the above type of chalcone derivatives, which can be rich source for further exploitation, can still give lead compounds.

REFERENCES:

1. Abdur Rahman, Rumana Qureshi, Mehvish kiran, Farzana Latif Ansari, Turk J , 31 2007; 25 –34.
2. Vogel’s Textbook of practical organic chemistry, fifth edition, by Funiss B.S., Hannford A. J., Smith P.W.G., Tatchell A.R., (2004),1032-1035.
3. Advanced Organic Chemistry, Reactions, Mechanisms, and Structure, Fourth edition,by Jerry March, A Wiley-Interscience Publication 939-943.
4. Morrison and Boyd, Organic Chemistry, sixth edition, 2004, 971-990, 997-820.
5. Bhardwaj SD, Jolly VS. Asian J Chem.1997; 9:48.
6. Desai JM, Shah VH. Indian J Chem. 2003; 42(B):631.
7. Tirlapur VK and Tadmalle T. “Synthesis and insecticidal activity of 1,2, 4triazothiazolidin-4-one derivatives.”Der Pharmacia Sinica 2011; 2 (1): 135-141.
8. Hussain s, SharmaJ and Amir M. “Synthesis and Antimicrobial Activities of 1,2,4-Triazol and 1,3,4-Thiadiazole Derivatives of 5-Amino-2-Hydroxybenzoic Acid.” E-Journal of Chemistry 2008; 5(4): 963-968.
   

   ---

   How to cite this article:

   Akram M, Sayeed A and Hussain SY: Synthesis and biological activities of some new heterocycles containing nitrogen and sulphur. Int J Pharm Sci Res 2017; 8(2): 865-77.doi: 10.13040/IJPSR.0975-8232.8(2).865-77.

   ---

   All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

   ---